Unregulated Elevation of Glutamate Dehydrogenase Activity Induces Glutamine-Stimulated Insulin Secretion: Identification and Characterization of a GLUD1 Gene Mutation and Insulin Secretion Studies With MIN6 Cells Overexpressing the Mutant Glutamate Dehydrogenase

The 12 patients with PHHI were part of a cohort we previously studied (9). Patients with mutations in the SUR1 gene were excluded, and none of the 12 had mutations of the Kir6.2 gene. No information was available regarding plasma ammonium levels for these patients at the initiation of this study. Patients with type 2 diabetes and nondiabetic control subjects were recruited from Yamaguchi University Hospital and affiliated hospitals. At the time of recruitment, informed consent was obtained from each individual according to a protocol approved by the Human Studies Committee of Yamaguchi University School of Medicine, and the investigation was performed in accordance with the guidelines expressed in the Declaration of Helsinki.

All 13 coding exons of the human GLUD1 gene were examined by PCR/single-strand conformation polymorphism (SSCP) analysis. Each of the exons was PCR amplified from peripheral blood leukocyte DNA. Primers for exons 11 and 12 were synthesized according to Stanley et al. (3). Other primers were designed based on the public database sequences (10) (accession nos. X66300–X66309, X66311–X66319, and AL136982) (Table 1). SSCP analysis was performed at 15°C using a CleanGel DNA analysis kit and a Multiphor II electrophoresis system (Amersham Pharmacia Biotech, Tokyo) as previously described (9). Nucleotide changes were identified by direct sequencing of PCR products using a model 373A automated sequencer and BigDye terminators cycle sequencing kit (Applied Biosystems Japan, Tokyo).

The full coding region of human GDH cDNA was obtained from 293 cell RNA by RT-PCR using primers 5′-CGC TTG TGG CCA TGT ACC-3′ (87–104, Genbank accession no. M20867) and 5′-TGT AAA CTT GTG ATA GGT CTG CAG-3′ (1,843–1,820). The GDH cDNA was cloned in pcDNA3 (Invitrogen, Carlsbad, CA) to make pcDNA3-hGDH, and the sequence was confirmed. A mutant GDH266C cDNA was made using a GeneEditor in vitro site-directed mutagenesis system (Promega, Tokyo) with mutagenesis primer 5′-CTC TAT GAG ATg TTT ACA TCG TTT TGG-3′ and a bottom-select oligonucleotide that was supplied in the kit.

The plasmids pcDNA3-hGDH and pcDNA3-hGDH266C were introduced into COS-7 cells by transfection with TransIT LT1 (PanVera, Madison, WI) according to the supplier’s protocol. A reporter plasmid, pcDNA3-lacZ, was cotransfected. Forty-eight hours after the transfection, cells were collected, suspended in PBS, and disrupted by sonification. After a brief centrifugation, the supernatant was used for GDH enzyme assay. β-Galactosidase activity was also measured (11). In MIN6 cells, wild-type and mutant GDH were overexpressed using retrovirus-mediated gene transfer as described below. Samples for the GDH enzyme assay were prepared as for COS-7 cells.

GDH activity was measured as previously described (12) using a Beckman Coulter (Fullerton, CA) Spectrophotometer Model DU-640 at 25°C. The assay solution (1 ml) consisted of 10 mmol/l Tris-acetate (pH 8.0), 10 μmol/l EDTA, 100 μmol/l NADH, 50 mmol/l NH₄Cl, and 5 mmol/l α-ketoglutarate. ADP or GTP was added to the solution at various concentrations. The reaction was started by adding cell extracts, and the decrease in absorbance at 340 nm was measured for 5 min. The activity was determined in duplicate or in triplicate for each sample. The half-maximal stimulatory concentration (SC₅₀) and the half-maximal inhibitory concentration (IC₅₀) were determined graphically.

The mutant GDH266C cDNA was inserted into the retrovirus vector pMX-puro (kindly provided by Dr. T. Kitamura, Institute of Medical Science, University of Tokyo) to make pMX-puro-GDH266C. Another retrovirus vector, pMX-puro-lacZ, which encodes the bacterial β-galactosidase gene, was also constructed and used as a control. Recombinant retroviruses were produced by transfecting these recombinant retrovirus vectors into a transient retrovirus packaging cell line, Plat-E cells (13), using FuGENE 6 (Roche, Indianapolis, IN) according to the supplier’s protocol. Culture media containing infectious retrovirus particles were collected 48 h after transfection.

MIN6 cells (14) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, St Louis, MO) containing 25 mmol/l glucose and supplemented with 15% of heat-inactivated FCS, 72 μmol/l β-mercaptoethanol, 50 unit/ml penicillin G, and 50 μg/ml streptomycin (DMEM-MING). MIN6 cells were infected with the recombinant retroviruses by incubating the cells with media containing the retroviruses for 24 h in the presence of 10 μg/ml polybrene (Sigma). Seventy-two hours after the initiation of infection, puromycin (2 μg/ml) was added to the culture medium, and MIN6 cells integrating the retroviral genome (MIN6-GDH266C and MIN6-lacZ) were selected.

MIN6 cells overexpressing the mutant GDH via retrovirus-mediated gene transfer (MIN6-GDH266C) and control lacZ-overexpressing cells (MIN6-lacZ) were seeded onto 24-well plates at a concentration of 3.5 × 10⁵ cells/well and cultured in DMEM-MIN6 medium. Sixty hours later, insulin secretion was assayed by the static incubation method (15). In brief, after a 30-min preincubation in HEPES-balanced Krebs-Ringer bicarbonate buffer (KRBB) (10 mmol/l HEPES, 120 mmol/l NaCl, 4.7 mmol/l KCl, 1.2 mmol/l MgSO₄, 1.2 mmol/l KH₂PO₄, 20 mmol/l NaHCO₃, and 2 mmol/l CaCl₂, pH 7.4) supplemented with 0.5% BSA and 5 mmol/l glucose, the preincubation buffer was replaced with fresh HEPES-balanced KRBB containing 0.5% BSA and various concentrations of glutamine or glucose. After an additional 2-h incubation at 37°C, the buffer was collected, and immunoreactive insulin was measured by radioimmunoassay using rat insulin (Linco Research, St. Charles, MO) as a standard. The amounts of secreted insulin were corrected by the amounts of cell protein in each well.

Human mitochondrial GDH is encoded by 13 exons of the GLUD1 gene, which is located at 10q23.3. Several related pseudogenes (GLUDP1–5) have been recognized (10,16). Among them, the GLUDP2 and -3 genes contain three exons (exons 2–4) that are highly homologous to the GLUD1 gene (10). Due to the extensive similarity of intron sequences surrounding these exons among the GLUD1 gene and its pseudogenes, we carefully chose the primer sequences so that these GLUD1 gene exons could be specifically PCR amplified from genomic DNA (data not shown).

We identified three nucleotide substitutions in 12 Japanese patients with PHHI. Two of those were silent mutations: CTA (Leu) to CTG (Leu) at codon 261 and GTG (Val) to GTC (Val) at codon 496. Altogether, 5 of 12 PHHI patients (42%) and 50 of 94 control subjects (53%) were heterozygotes of CTA/CTG at codon 261. Although they were easily recognized on SSCP analysis and were confirmed by sequencing, the CTA/CTA homozygote and the CTG/CTG homozygote were barely distinguishable by SSCP patterns. Therefore, we did not determine the frequency of this single nucleotide polymorphism. One PHHI patient was a heterozygote at codon 496 (GTG/GTC). The frequency of this variation was not determined in control subjects. The third nucleotide variation, TAT to TGT at codon 266, was found in one patient with PHHI. This nucleotide substitution changes Tyr266 to Cys, and the patient was a heterozygote. This mutation was not found in 79 patients with type 2 diabetes or in 94 control subjects.

The female patient with the Y266C mutation was 21 years of age at the time of this study. She was born at 37 weeks and 5 days of gestation as the second of four daughters of nonconsanguineous parents. Pregnancy and delivery were normal. Her birth weight was 2,700 g. Her first seizure occurred at 7 months of age and was not responsive to anticonvulsants. Hypoglycemia (1.7 mmol/l) was disclosed at age 13 months, when a seizure occurred just after milk feeding. Oral administration of leucine (150 mg/kg) induced hypoglycemia, and diazoxide treatment was started under a diagnosis of leucine-sensitive hypoglycemia. After the initiation of diazoxide treatment, no hypoglycemic episodes were documented up to the time of this study, i.e., at 21 years of age. She has no family history of hypoglycemia, and her parents and three sisters were healthy. Her plasma ammonium levels, measured during this study, were mildly elevated (128 μmol/l, normal 12–84). Her parents and two of her sisters did not have the Y266C mutation, and their plasma glucose and ammonium levels were normal (data not shown).

The wild-type and mutant GDH were expressed in COS-7 cells by transfection of pcDNA3-hGDH and pcDNA3-hGDH266C, respectively. A reporter plasmid pcDNA3-lacZ was cotransfected. GDH activities in the crude extracts of COS-7 cells transfected with pcDNA3-hGDH and pcDNA3-hGDH266C were 0.82 ± 0.22 and 3.27 ± 0.72 μmol · NADH · mg^–1 · protein · min^–1, respectively, when activity was measured without ADP or GTP in the reaction mixture (means ± SE, n = 3). Endogenous GDH activity in untreated COS-7 cells (0.028 μmol/l · NADH · mg^–1 · protein · min^–1) was <1/20 the total GDH activity in COS-7 cells transfected with pcDNA3-hGDH and thus negligible compared with the exogenous GDH expressed by the transfected plasmids. Therefore, the basal activity (activity in the absence of ADP or GTP) of GDH266C is elevated as compared with the wild-type enzyme. To exclude the possibility that this difference in the enzyme activity reflected the difference in the transfection efficiency, we measured β-galactosidase activities in the same cell extracts, and the GDH activity was normalized by the β-galactosidase activities. After the normalization, the basal activity of GDH266C was still 3.5 ± 1.2 times higher (means ± SE, n = 3) than that of wild-type GDH.

GDH is known to be regulated by various allosteric regulators. We examined whether the activity of GDH266C can be regulated by ADP, an allosteric activator, or GTP, an allosteric inhibitor. As shown in Fig. 1, ADP activated wild-type GDH (3.32 ± 0.12 μmol/l · NADH · mg^–1 · protein · min^–1 at 200 μmol/l ADP), with a SC₅₀ of 11 μmol/l. On the other hand, the activity of GDH266C was not increased by ADP (3.45 ± 0.50 μmol/l · NADH · mg^–1 · protein · min^–1 at 200 μmol/l ADP). Similarly, although GTP inhibited the wild-type GDH activity, with a IC₅₀ of 0.65 μmol/l, the activity of GDH266C was not inhibited by GTP at a concentration as high as 100 μmol/l. The activities of wild-type GDH and GDH266C at 100 μmol/l GTP were 2.7 ± 0.9 and 96.6 ± 1.6% of those without GTP, respectively.

We then overexpressed GDH266C in an insulinoma cell line, MIN6. Using a retrovirus-mediated gene transfer system, the mutant GDH gene was transferred to MIN6 cells along with the puromycin resistance gene. The cells stably integrating the retroviral genome were chosen by puromycin selection. MIN6 cells overexpressing bacterial β-galactosidase (MIN6-lacZ) were also established and used as a control.

Determined in crude cell extracts (Table 2), basal (in the absence of ADP) and inhibited (in the presence of GTP) GDH activities in MIN6-GDH266C were 20 times higher than the basal GDH activity in MIN6-lacZ. The values were even higher than activated MIN6-lacZ GDH activity (measured in the presence of 200 μmol/l ADP). GDH activity in MIN6-GDH266C was doubled in the presence of 200 μmol/l ADP, probably because intrinsic MIN6 GDH was activated. These results suggest that in MIN6-GDH266C, GDH activity is constitutively elevated and minimally regulated by allosteric regulators such as ADP and GTP.

Insulin secretion was analyzed in these cells. Glutamine, which is permeable to the plasma membrane and converted to glutamate inside β-cells, did not stimulate insulin secretion from MIN6-lacZ cells, as was reported for intact islets (7). On the other hand, it remarkably stimulated insulin secretion from MIN6-GDH266C in a dose-dependent manner (Fig. 2A). Glucose-stimulated insulin secretion was also studied. In MIN6-GDH266C, insulin secretion was significantly exaggerated at low glucose concentrations (2 and 5 mmol/l; P = 0.009 at 2 mmol/l and P = 0.020 at 5 mmol/l, unpaired Student’s t test), as compared with control MIN6-lacZ, but not at higher glucose concentrations (8–25 mmol/l) (Fig. 2B).

We found one patient with the GLUD1 gene mutation, Y266C, in a cohort of 12 patients with PHHI. The patient was heterozygous for the mutation and had clinical features of HI/HA syndrome (3–5). The Y266C mutation appeared to be a de novo mutation in this patient. The same mutation was recently described in a sporadic HI/HA syndrome patient (5), although the kinetic properties of the mutant enzyme have not been studied. It has been reported that Y262 of bovine GDH, corresponding to Y266 of the human enzyme, is in the GTP binding pocket and interacts with the γ-phosphate moiety of GTP (5,17–19). Chemical modification of this residue resulted in reduced sensitivity to GTP inhibition and ADP activation (20). The reduced sensitivity to ADP activation is probably attributable to a secondary effect of modification of the GTP binding site because the affinity of ADP for the chemically modified GDH was not altered (20). We expressed GDH266C in COS-7 cells and examined the effect of this mutation on kinetic properties. Strikingly, the allosteric regulation of GDH266C by GTP and ADP was more severely impaired than that of the enzyme in which the corresponding amino acid was chemically modified. GDH266C was essentially insensitive to GTP inhibition or ADP activation (Fig. 1). Basal GDH266C activity, measured in the absence of GTP or ADP, appeared to be higher than that of the wild-type GDH when it was estimated in crude cell extracts (see results). Although quantitative evaluation of kinetic properties requires studies using purified enzyme rather than analysis in the crude cell extract, GDH266C appeares to be a constitutively activated enzyme.

The nearly total insensitivity of GDH266C to GTP and ADP contrasts with the kinetic characteristics of other mutants that were recently identified in patients with HI/HA syndrome. In previous studies, the IC₅₀ elevation for GTP was 2- to 10-fold, with the IC₅₀ being <2 μmol/l (3–5), and activation by ADP was mostly intact. The difference is probably attributable to the nature of the mutations. In addition, it may also be explained by the fact that the previous studies were performed using patients’ lymphoblasts in which both mutant and wild-type GDH existed at a 1:1 ratio, and therefore the effects of mutations on kinetic properties were less prominent.

Despite the differences in the kinetic properties of the enzyme, the clinical characteristics of our patient and the previously reported patient with the same mutation (5) were very similar to those of patients with other mutations. It was recently reported that there is a genotype-phenotype correlation in regard to plasma ammonium levels but not the severity of hypoglycemia (5). Hypoglycemia in our patient was medically manageable with diazoxide, and the plasma ammonium level was only mildly elevated (128 μmol/l, normal 12–84).

Using GDH266C, which has unique kinetic properties, as a tool, we examined the role of GDH in the regulation of insulin secretion and the pathophysiology of β-cells from HI/HA syndrome patients. We established MIN6 cells stably overexpressing the mutant GDH and analyzed glutamine- and glucose-stimulated insulin secretion. GDH, upon stimulation by allosteric activators such as leucine, presumably enhances glutamate oxidation and thereby stimulates insulin secretion (1,2). Herein, we have directly shown for the first time that increased GDH activity confers on β-cells the ability to secrete insulin in response to glutamine. GDH266C-overexpressing MIN6 cells secreted insulin in response to glutamine in a dose-dependent manner, whereas control β-galactosidase–overexpressing MIN6 cells were unresponsive to glutamine stimulation. This observation clearly demonstrated the importance of GDH catalyzing oxidative deamination of glutamate for insulin secretion rather than glutamate formation from α-ketoglutarate by reverse reaction (6). Enhanced insulin secretion in response to glutamine from MIN6-GDH266C is directly relevant to the phenotype of HI/HA syndrome patients—they tend to develop hypoglycemia after a high-protein meal (21).

Physiologically, glutaminolysis may play more important roles in insulin secretion at low glucose concentrations and contribute to interprandial, basal insulin secretion (8). In pancreatic islets, glucose increases GTP levels while decreasing ADP levels in a concentration-dependent manner (22–24). At low glucose concentrations, flux through the TCA cycle via glucose metabolism is low, whereas glutaminolysis is activated because relatively low intracellular GTP concentrations and high ADP concentrations activate GDH, leading to sustained basal insulin secretion (8). In MIN6-GDH266C, in which GDH activity is supraphysiologically elevated, insulin secretion was exaggerated significantly at low glucose concentrations (2 and 5 mmol/l) compared with control MIN6-lacZ. This observation is consistent with a previous study (8) and may also explain the HI/HA syndrome patients’ fasting hypoglycemia. At higher glucose concentrations, flux through the TCA cycle is probably high enough to mask the effect of enhanced glutaminolysis on insulin secretion in MIN6-GDH266C cells.

Recently, glutamate was suggested to be a mediator of glucose-stimulated insulin secretion, acting directly on insulin secretory granules (6). According to this hypothesis, GDH catalyzes the formation of glutamate from α-ketoglutarate rather than oxidative deamination of glutamate. However, our results from MIN6-GDH266C and previous evidence, including clinical data from HI/HA syndrome, do not support this hypothesis (3–5,7,8). Investigations to elucidate changes in intracellular glutamate concentrations and glutamine oxidation in MIN6-GDH266C will further clarify the role of GDH in the regulation of insulin secretion.

This study was partly supported by Grants-in-Aid for Scientific Research (12671113 and 11557012 to Y.T.) and for Creative Basic Research (10NP0201 to Y.O.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

We thank all of our patients, their family members, and physicians caring for the patients who participated in this study. We also thank Dr. T. Kitamura (Institute of Medical Science, University of Tokyo) for providing us with the retrovirus vector pMX-puro and the transient retrovirus packaging cell line, Plat-E, as well as Yukari Kora-Miura and Atsuko Tanimura for their expert technical assistance. We are grateful to Dr. K. Ariyoshi (Yamaguchi University) for his help in preparing recombinant retroviruses.